WO2001064912A2 - Enzymes de degradation de triazine - Google Patents

Enzymes de degradation de triazine Download PDF

Info

Publication number
WO2001064912A2
WO2001064912A2 PCT/US2001/006654 US0106654W WO0164912A2 WO 2001064912 A2 WO2001064912 A2 WO 2001064912A2 US 0106654 W US0106654 W US 0106654W WO 0164912 A2 WO0164912 A2 WO 0164912A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
seq
nucleic acid
sequence
triazine
Prior art date
Application number
PCT/US2001/006654
Other languages
English (en)
Other versions
WO2001064912A3 (fr
Inventor
Sun Ai Raillard
Jeremy Minshull
Claes Gustafsson
Original Assignee
Maxygen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Maxygen, Inc. filed Critical Maxygen, Inc.
Priority to EP01916339A priority Critical patent/EP1268815A2/fr
Priority to AU2001243376A priority patent/AU2001243376A1/en
Publication of WO2001064912A2 publication Critical patent/WO2001064912A2/fr
Publication of WO2001064912A3 publication Critical patent/WO2001064912A3/fr

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)

Definitions

  • Atrazine and other triazine derivatives are widely used as herbicides for broad leaf weed control. Approximately 800 million pounds of these compounds were used in the U.S. between 1980 and 1990. As a result of this widespread use, the compounds have been detected in ground and surface water in the U.S. and in many other countries that use triazine derivatives.
  • Atrazine and related compounds are degraded very slowly in nature.
  • atrazine has a water solubility of 33 mg/liter at 27°C and its half -life can vary from about 4 to about 60 weeks when present in soil. Therefore, high concentrations of triazine derivatives in soil can take quite a long time to dissipate.
  • Isolation of triazine and/or atrazine degrading microorganisms has been reported by numerous sources. See, e.g., Behki et al., J. Agric. Food Chem. 34, 746-749 (1986); Behki et al., Appl. Environ. Microbiol. 59, 1955-1959 (1993); Cook, FEMS Microbiol. Rev. 46. 93-116 (1987); Cook et al.. J. Agric. Food Chem. 29, 1135-1143 (1981); Erikson et al., Critical Rev. Environ. Cont. 19, 1-13 (1989);
  • Atrazine degrading enzymes e.g., atrazine chlorohydrolases
  • atrazine chlorohydrolases Genes encoding atrazine degrading enzymes, e.g., atrazine chlorohydrolases, have also been isolated. See, e.g., Souza et al., Appl. Environ. Microbiol. 61, 3373-3378 (1995). While this protein is useful for dechlorinating atrazine, improved triazine hydrolases are desirable.
  • the present invention provides novel triazine hydrolases that provide novel enzyme substrate activity. These hydrolases are useful in a variety of soil and water treatments and other industrial and commercial applications that will be apparent upon further review.
  • the present invention provides novel triazine hydrolases with improved characteristics such as activity against a wider range of substrates than wild type triazine hydrolases.
  • the invention provides isolated and recombinant nucleic acids corresponding to polynucleotides that are novel triazine hydrolases, encode novel triazine hydrolase proteins, hybridize under highly stringent conditions to such novel triazine hydrolases or polynucleotides encoding novel triazine hydrolase proteins, or fragments thereof encoding polypeptides with triazine hydrolase activity.
  • the invention provides polynucleotides which include a subsequence corresponding to one or more of SEQ ID NO:l to SEQ J-D NO: 48 or a complementary polynucleotide sequence thereof.
  • Polynucleotide sequences encoding a polypeptide selected from SEQ J-D NO: 49 to SEQ ID NO: 608, or a complementary polynucleotide sequence thereof are also provided.
  • Polynucleotide sequences which hybridize under highly stringent conditions over substantially the entire length of one or more of the above polynucleotide sequences provide additional embodiments.
  • Other embodiments include fragments of the above sequences, which fragments typically have triazine hydrolase activity.
  • the nucleic acids of the invention comprise a polynucleotide that encodes a hydrolase, e.g., a triazine hydrolase.
  • the triazine hydrolase typically hydrolyzes one or more of: aminoatrazine, atrazine, triazine, atratone, N-methylatrazine, ametryn, aminopropazine, propazine, prometon, N- methylpropazine, prometryn, aminomorphazine, morphazine, morphatryn, morphaton, or N-methylmorphazine.
  • the encoded polypeptide is typically about 450 to about 500 amino acids in length or about 474 amino acids in length.
  • Polypeptides comprising at least about 20 contiguous amino acids, at least about 50 contiguous amino acids, at least about 100 contiguous amino acids, or at least 150 contiguous amino acids of any one of SEQ ID NO: 49-608 are also provided.
  • the invention provides a cell comprising any of the nucleic acids described above.
  • Such cells typically express a polypeptide encoded by one of the nucleic acids of the invention.
  • vectors comprising the nucleic acids of the invention are provided.
  • the vector e.g., an expression vector, typically comprises a plasmid, a cosmid, a phage, a virus, or the like. Cells transduced by such vectors are also provided.
  • the invention provides remediation compositions comprising a cell comprising the polypeptides or polynucleotides of the invention.
  • the remediation compositions are typically used to treat or decontaminate triazine or atrazine contaminated water, soil, or the like.
  • Such remediation compositions are optionally used in the methods of the invention. For example, a method of treating a sample comprising atrazine or a triazine derivative is provided.
  • the method comprises adding a composition to a sample comprising atrazine or a triazine derivative, wherein the composition comprises a polypeptide encoded by a nucleic acid of the invention.
  • the nucleic acids and polypeptides of the invention are thus used to decontaminate triazine contaminated soil, water, or the like.
  • Compositions containing two or more nucleic acids of the invention are an additional feature of the invention. In some cases, these compositions are libraries of nucleic acids, preferably containing at least ten such nucleic acids.
  • compositions produced by digesting one or more nucleic acids of the invention are also a feature of the invention, as are compositions produced by incubating one or more nucleic acids of the invention, e.g., in the presence of deoxyribonucleotide triphosphates and a nucleic acid polymerase, such as a thermostable polymerase.
  • Isolated or recombinant polypeptides encoded by the nucleic acids of the invention are also provided. For example, polypeptides comprising a sequence selected from SEQ ID NO: 49-608 are provided.
  • polypeptides typically have triazine hydrolase activity of at least 50,000 nM per hour or about 2-fold to at least about 200-fold greater than an atrazine chlorohydrolase corresponding to U55933.
  • Polypeptides comprising about 100 contiguous amino acids, at least about 150 contiguous amino acids of the encoded protein, or at least about 250 contiguous amino acids of the encoded protein are also provided.
  • polypeptides of the invention with secretion/localization sequences are a feature of the invention, as are polypeptides with purification subsequences, including epitope tags, FLAG tags, polyhistidine tags, GST fusions, and the like.
  • polypeptides of the invention bearing a methionine at the N- terminus or comprising one or more modified amino acid e.g., a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, or the like, are features of the invention.
  • the invention provides methods of producing polypeptides.
  • the methods typically comprise introducing into a population of cells a nucleic acid or recombinant expression vector of the invention.
  • the nucleic acid is generally operatively linked to a regulatory sequence effective to produce the encoded polypeptide.
  • the cells are cultured in a culture medium to produce the polypeptide, which is isolated from the cells or from the culture medium.
  • Another aspect of the invention relates to DNA shuffling to provide novel triazine hydrolase homologues by recursively recombining one or more nucleic acid of the invention with one or more additional nucleic acid, such as a nucleic acid encoding a triazine hydrolase homologue or subsequence thereof.
  • recursive recombination produces at least one library of recombinant triazine hydrolase homologue nucleic acids.
  • the libraries so produced are embodiments of the invention as are cells comprising the libraries.
  • methods of producing modified triazine hydrolases by mutating the nucleic acids of the invention are provided.
  • nucleic acids comprising unique subsequences selected from SEQ JJD NO: 1 to SEQ ID NO: 48, ( as compared to a nucleic acid corresponding to U55933); polypeptides comprising a unique subsequence from: SEQ JJD NO: 49- 608, (as compared to a polypeptide corresponding to U55933); and target nucleic acids that hybridize under stringent conditions to a unique coding oligonucleotide that encodes a unique subsequence of a polypeptide selected from: SEQ ID NO: 49-608, (unique as compared to a polypeptide corresponding to U55933) are also features of the invention.
  • the invention also provides computers, computer readable mediums and integrated systems, including databases that are composed of sequence records including character strings corresponding to SEQ JD NO:l to SEQ ID NO: 608.
  • integrated systems optionally include one or more instruction set for selecting, aligning, translating, reverse-translating, or viewing any of the above character strings with each other and/or with any additional nucleic acid or amino acid sequence.
  • Figure 1 illustrates the conversion of atrazine to hydroxyatrazine via dechlorination catalyzed by atrazine chlorohydrolase.
  • Figure 2 defines and illustrates a variety of triazine derivatives designed to explore chemical space.
  • One side chain is fixed as isopropyl amine and the second side chain (Rl) and the leaving group (R2) are varied, e.g., to confer increased bulkiness.
  • Figure 3 provides turnover rates in nM substrate/h/20 ⁇ l cells
  • FIG. 4 provides a distribution of functional activity in sequence space. Triazine hydrolase activities towards each of 15 substrates is indicated by a circle whose area is proportional to the activity. Substrates are arranged in a grid format as in Figure 2. Activities are shown on a phylogenetic tree to show relationships between enzyme sequences.
  • Atrazine and other triazine derivatives are widely used in herbicides, either alone or in combination with other compounds, e.g., for control of broad-leaf weeds. Atrazine and triazine runoff and persistence in soil and water often lead to triazine levels exceeding EPA limits for drinking water. Solutions to these concerns, e.g., atrazine contaminated soil, involve introduction of indigenous and/or recombinant bacteria to metabolize or degrade triazine compounds in soil or water. Wild-type hydrolases present in indigenous bacteria degrade atrazine at low levels but do not degrade or metabolize other triazine derivatives. Figure 1 illustrates the conversion of atrazine to hydroxyatrazine using atrazine chlorohydrolase.
  • Triazine hydrolase refers to an enzyme or polypeptide having triazine hydrolase activity, i.e., the ability to hydrolyze or otherwise degrade one or more species of the class of compounds represented by:
  • Ri and R 3 each independently comprise an amino group, i.e., -NH 2 , or a substituted linear, branched, or cyclic amino group.
  • Ri and R 3 are each independently a lower-alkyl-substituted amino group or a morpholino group.
  • the term "lower alkyl” refers to a C ⁇ - 6 alkyl. More typically, Ri and R are each independently -NH(C 2 H 5 ), -NHCH(CH 3 ) 2 ,
  • R 3 is -NHCH(CH 3 ) 2
  • R 2 is an amino group, i.e., -NH 2> or an optionally substituted amino group, e.g., -NRH or -NRR', a halo, a lower alkoxy, or S-R, where R and R' are each independently a lower alkyl group.
  • R 2 is - NH 2 , -X, wherein X is a halogen such as Cl, -OCH 3 , -NH(CH 3 ), or -S-CH 3 .
  • Figure 2 defines the R groups for a variety of triazine compounds.
  • a "polynucleotide sequence” is a nucleic acid (which is a polymer of nucleotides (A,C,T,U,G, etc. or naturally occurring or artificial nucleotide analogues) or a character string representing a nucleic acid, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.
  • an "amino acid sequence” is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.
  • a nucleic acid, protein or other component is “isolated” when it is partially or completely separated from components with which it is normally associated (other proteins, nucleic acids, cells, synthetic reagents, etc.).
  • a nucleic acid or polypeptide is "recombinant” when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid.
  • a “subsequence” or “fragment” is any portion of an entire sequence, up to and including the complete sequence.
  • Numbering of a given amino acid or nucleotide polymer “corresponds to numbering" of a selected amino acid polymer or nucleic acid when the position of any given polymer component (amino acid residue, incorporated nucleotide, etc.) is designated by reference to the same residue position in the selected amino acid or nucleotide, rather than by the actual position of the component in the given polymer.
  • a vector is a composition for facilitating cell transduction by a selected nucleic acid, or expression of the nucleic acid in the cell.
  • Vectors include, e.g., plasmids, cosmids, viruses, YACs, bacteria, poly-lysine, etc.
  • Substantially an entire length of a polynucleotide or amino acid sequence refers to at least about 70%, generally at least about 80%, or typically ' about 90% or more of a sequence.
  • an “antibody” refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of irnmunoglobulin genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
  • Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond.
  • the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. Fourth Edition (1998), for a more detailed description of other antibody fragments).
  • Antibodies include single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
  • sFv single chain Fv
  • the invention provides isolated or recombinant atrazine hydrolase polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides.
  • a small library of triazine hydrolases i.e., about 1500 clones, were screened by high throughput mass spectrometry (See, e.g., High Throughput Mass Spectrometry, By
  • 029510US for methods of performing high-throughput mass spectrometry) for activity against about 15 different triazine derivatives, covering a chemical structure space enabling investigation of leaving group chemistry as well as substrate bulkiness
  • the proteins of the present invention were screened for hydrolase activity against atrazine and other triazine derivatives as represented by ⁇
  • triazine compounds include, but are not limited to aminotriazine, atrazine, atratone, N-methylatrazine, ametryn, aminopropazine, propazine, prometon, N-methylpropazine, prometryn, aminomorphazine, morphazine, morphatryn, morphaton, and N-memylmorphazine, which are all depicted and defined in Figure 2.
  • Clones were identified that showed about 2-fold to about 180 or 200-fold improvement over a wild type atrazine chlorohydrolase (as characterized at GenBank #U55933).
  • the hydrolases can display an improvement of about 500-fold or more.
  • Modified amino acid at positions 84 and 92 can affect the bulk of side chain Rl that can be accepted.
  • a novel triazine hydrolase was created with an alanine at position 92 in combination with asparagine and serine at positions 328 and 331, respectively. This novel hydrolase showed enhanced dechlorination activity versus bulkier substrates.
  • Novel triazine hydrolases were found that that yielded a higher transformation rate, e.g., up to 150-fold greater, than native hydrolases.
  • the novel triazine hydrolases provided herein also hydrolyzed at least five triazine compounds, e.g., prometon, prometryn, N-methylaminopropazine, morphazine, and aminomorphazine, that were not hydrolyzed by a native hydrolase.
  • FIG 4 provides a distribution of functional activity ion sequence space. Triazine hydrolase activity toward each of the 15 substrates provided in Figure 2 is indicated by a circle whose area is proportional to the activity. The 15 substrates are arranged in a grid that shows relationships between enzyme sequences.
  • Exemplary nucleic acids that encode polypeptides having improved or expanded triazine degradation properties, such as degradation activity with respect to a variety of triazine derivatives, are provided in SEQ ID NO: 1 to SEQ JD NO: 48 encoding the polypeptides provided in SEQ ID NO: 49 to SEQ ID NO: 96.
  • Additional triazine degrading polypeptides are exemplified by SEQ JJD NOs: 97-608, as shown in Table 5.
  • the sequences listed in Table 5, e.g., SEQ JJD NOs: 97-608, are based on the polypeptide sequence of atrazine hydrolase (atzA) (Genbank #U55933), with corresponding numbering.
  • SEQ DD NO: 97 comprises a phenylalanine at position 84, a leucine at position 92, an aspartic acid residue at position number 125, an isoleucine at position number 217, a proline residue at position 219, an isoleucine at position number 253, a glycine at position 255, an aspartic acid at position 328, and a cysteine at position 331.
  • Polynucleotides and oligonucleotides of the invention can be prepared by standard solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated recombination methods) to form essentially any desired continuous sequence.
  • the polynucleotides and oligonucleotides of the invention can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage et al, (1981) Tetrahedron Letters 22:1859-69, or the method described by Matthes et al, (1984) EMBO J.
  • oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others.
  • peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc., and many others.
  • Certain polynucleotides of the invention may also obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify polynucleotides which encode the triazine hydrolase polypeptides and fragments of those polypeptides.
  • Procedures for screening and isolating cDNA clones are well-known to those of skill in the art. Such techniques are described in, for example, Sambrook et al. (1989) supra, and Ausubel FM et al (1989; supplemented through 1999) supra.
  • the polynucleotides of the invention include sequences which encode novel triazine hydrolases and sequences complementary to the coding sequences, and novel fragments of coding sequence and complements thereof.
  • the polynucleotides can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, synthetic RNA and DNA, and cDNA.
  • the polynucleotides can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (anti-sense, complementary) strand.
  • the polynucleotides optionally include the coding sequence of a triazine hydrolase (i) in isolation, (ii) in combination with additional coding sequence(s), so as to encode, e.g., a fusion protein, a pre-protein, a prepro-protein, or the like, (iii) in combination with non-coding sequences, such as introns, control elements such as a promoter, a terminator element, or 5' and/or 3' untranslated regions effective for expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment in which the triazine hydrolase coding sequence is a heterologous gene. Sequences can also be found in combination with typical compositional formulations of nucleic acids, including in the presence of carriers, buffers, adjuvants, excipients, vectors, vector components, and the like.
  • the polynucleotides of the invention have a variety of uses in, for example: recombinant production (i.e., expression) of the triazine hydrolase polypeptides of the invention; as soil or water treatment compositions, e.g., to encode enzymes which degrade triazine, atrazine or other triazine derivatives; as diagnostic probes for the presence of complementary or partially complementary nucleic acids
  • triazine hydrolase polypeptides or "triazine hydrolases” are used in recombinant DNA molecules that direct the expression of the triazine hydrolase polypeptides in appropriate host cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence are also used to clone and express the triazine hydrolases.
  • a coding sequence can be modified to enhance its expression in a particular host.
  • the genetic code is redundant, with 64 possible codons, but most organisms preferentially use a subset of these codons.
  • the codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons (see, e.g., Zhang SP et al. (1991) Gene 105:61-72). Codons can be substituted to reflect the preferred codon usage of the host, a process called "codon optimization" or "controlling for species codon bias.”
  • Optimized coding sequence containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half -life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for S. cerevisiae and mammals are UAA and UGA respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E. coli prefer to use UAA as the stop codon (Dalphin ME et al. (1996) Nuc. Acids Res. 24: 216-218).
  • polynucleotide sequences of the present invention are optionally engineered in order to alter a triazine hydrolase coding sequence, for a variety of reasons, including, but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
  • alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis or de novo synthesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.
  • the present invention also includes recombinant constructs comprising one or more of the nucleic acid sequences as broadly described above.
  • the constructs comprise a vector, such as, a plasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), an agrobacterium, or the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • RNA polymerase mediated techniques e.g., NASBA
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA RNA polymerase mediated techniques
  • the present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the triazine hydrolase gene.
  • the culture conditions are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited above. Additional useful references for cloning and culture of cells include, including, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York and the references cited therein, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY, and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
  • the triazine hydrolase proteins of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like.
  • bacteria expressing the polypeptides of the invention are optionally used to degrade triazine compounds, e.g., in triazine contaminated water or soil.
  • Gamborg and Phillips eds (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York).
  • the polynucleotides of the present invention may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses, agrobacterium, and many others.
  • the nucleic acid sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis.
  • promoters include: LTR or S V40 promoter, E. coli lac or tip promoter, phage lambda P promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
  • the vector optionally includes appropriate sequences for amplifying expression.
  • the expression vectors optionally comprise one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. Examples of appropriate expression hosts especially include bacterial cells, such as E.
  • a number of expression vectors may be selected depending upon the use intended for the triazine hydrolase. For example, when large quantities of triazine hydrolase or fragments thereof are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable. Such vectors include, but are not limited to, multifunctional E.
  • coli cloning and expression vectors such as BLUESCRJPT (Stratagene), in which the triazine hydrolase coding sequence is optionally ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pTN vectors (Van Heeke & Schuster (1989) / Biol Chem 264:5503-5509); pET vectors (Novagen, Madison WI); and the like.
  • BLUESCRJPT Stratagene
  • pTN vectors Vectors (Van Heeke & Schuster (1989) / Biol Chem 264:5503-5509)
  • pET vectors Novagen, Madison WI
  • chimeric nucleic acids or other sequences are introduced into the cells of particular organisms of interest.
  • bacterial cells any of which may be used in the present invention. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment, and infection with viral vectors, etc.
  • Bacterial cells can be used to amplify the number of plasmids containing DNA constructs of this invention.
  • Bacteria are typically grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook).
  • kits are commercially available for the purification of plasmids from bacteria. For their proper use, follow the manufacturer's instructions (see, for example, EasyPrepTM, FlexiPrepTM, both from Pharmacia Biotech; StrataCleanTM, from Stratagene; and, QIAexpress Expression SystemTM from Qiagen).
  • the isolated and purified plasmids are then further manipulated to produce other plasmids.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, et al, Nature, 328:731 (1987); Schneider, B., et al, Protein Expr. Purif.
  • Specific initiation signals can aid in efficient translation of a triazine hydrolase coding sequence. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where a triazine hydrolase coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon can be provided. The initiation codon is provided in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic.
  • Polynucleotides of the invention can also be fused, for example, in- frame to a nucleic acid encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle, or to direct polypeptide secretion to the periplasmic space or into the cell culture media.
  • sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a eukaryotic cell, such as a mammalian cell, a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • bacterial cells are often prefe ⁇ ed.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, or other common techniques (See, e.g., Sambrook, Ausubel, Berger (all supra). See also, Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology).
  • a host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post- translational processing which cleaves a "pre” or a "prepro” form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the co ⁇ ect modification and processing of the introduced, foreign protein.
  • stable expression For long-term, high-yield production of recombinant proteins, stable expression can be used.
  • cell lines which stably express a polypeptide of the invention are transduced using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene.
  • cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. For example, resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides encoding triazine hydrolases of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane, e.g., for use in bioremediation.
  • the polynucleotides of the present invention may also comprise a coding sequence fused in-frame to a marker sequence which, e.g., facilitates purification of the encoded polypeptide.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (co ⁇ esponding to an epitope derived from the influenza hemagglutinin protein; Wilson, I., et al. (1984) Cell
  • maltose binding protein sequences the FLAG epitope utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, WA), and the like.
  • the inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and the triazine hydrolase sequence is useful to facilitate purification.
  • One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al. (1992) Protein Expression and Purification 3:263-281) while the enterokinase cleavage site provides a means for separating the triazine hydrolase polypeptide from the fusion protein.
  • pGEX vectors Promega; Madison, WI
  • GST glutathione S-transf erase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
  • ligand-agarose beads e.g., glutathione-agarose in the case of GST-fusions
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
  • many references are available for the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archebacterial origin.
  • Polypeptides of the invention can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the tagging systems noted herein), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps.
  • HPLC high performance liquid chromatography
  • Cell-free transcription/translation systems can also be employed to produce polypeptides using DNAs or RNAs of the present invention. Several such systems are commercially available. A general guide to in vitro transcription and translation protocols is found in Tyrnms (1995) In vitro Transcription and Translation Protocols: Methods in Molecular Biology Volume 37, Garland Publishing, NY.
  • Polypeptides of the invention may contain one or more modified amino acid.
  • the presence of modified amino acids may be advantageous in, for example, (a) increasing polypeptide serum half-life, (b) reducing polypeptide antigenicity, and (c) increasing polypeptide storage stability.
  • Amino acid(s) are modified, for example, co- translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means.
  • Non-limiting examples of a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like.
  • polynucleotides also refe ⁇ ed to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least about 20, about 30, about 50 bases, or about 75 bases or more, which hybridize under highly stringent conditions to a triazine hydrolase polynucleotide as described above.
  • the polynucleotides are optionally used as probes, primers, sense and antisense agents, and the like, according to methods as noted supra.
  • nucleic acid sequences encoding triazine hydrolase polypeptides of the invention are optionally produced, some of which may bear minimal sequence homology to the nucleic acid sequences explicitly disclosed herein.
  • silent variations are one species of “conservatively modified variations", discussed below.
  • each codon in a nucleic acid can be modified by standard techniques to encode a functionally identical polypeptide.
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence.
  • the invention provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (e.g., as set forth in Table 1) as applied to the nucleic acid sequence encoding a triazine hydrolase polypeptide of the invention. All such variations of every nucleic acid herein are specifically provided and described by consideration of the sequence in combination with the genetic code.
  • Constants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
  • substitutions, deletions, or additions which alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are
  • “conservatively substituted variations” of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.
  • a conservatively substituted variation of the polypeptide identified herein as SEQ JJD NO:49 will contain "conservative substitutions", according to the six groups defined above, in up to 23 residues (i.e., 5% of the amino acids) in the 474 amino acid polypeptide.
  • NQI LLR GGP SHG examples include: NNI L K GGP AHG and
  • nucleic acid constructs which are disclosed yield a functionally identical construct.
  • substitutions i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
  • conserve amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct. Such conservative variations of each disclosed sequence are a feature of the present invention.
  • Nucleic Acid Hybridization Nucleic acids "hybridize" when they associate, typically in solution.
  • nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993)
  • highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
  • An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, supra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal. An example low stringency wash is 2x SSC at 40°C for 15 minutes. In general, a signal to noise ratio of 5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Comparative hybridization can be used to identify nucleic acids of the invention, and this comparative hybridization method is a prefe ⁇ ed method of distinguishing nucleic acids of the invention.
  • detection of highly stringent hybridization in the context of the present invention indicates strong structural similarity to, e.g., the nucleic acids provided in the sequence listing herein.
  • One measure of stringent hybridization is the ability to hybridize to one of the listed nucleic acids, e.g., nucleic acid sequences SEQ ID NO: 1 to SEQ JJD NO:48 and complementary polynucleotide sequences thereof, under highly stringent conditions. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid.
  • the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and or increasing the concentration of organic solvents such as formalin in the hybridization or wash), until a selected set of criteria are met.
  • the hybridization and wash conditions are gradually increased until a probe comprising one or more nucleic acid sequences selected from SEQ J-D NO:l to SEQ JD NO:48 and complementary polynucleotide sequences thereof, binds to a perfectly matched complementary target (again, a nucleic acid comprising one or more nucleic acid sequences selected from SEQ JJD NO:l to SEQ JJD NO:48 and complementary polynucleotide sequences thereof), with a signal to noise ratio that is at least 5x as high as that observed for hybridization of the probe to an unmatched target.
  • the unmatched target is a nucleic acid co ⁇ esponding to a known triazine hydrolase, e.g., a triazine hydrolase nucleic acid (other than those in the accompanying sequence listing) that is present in a public database such as GenBankTM at the time of filing of the subject application.
  • a known triazine hydrolase e.g., a triazine hydrolase nucleic acid (other than those in the accompanying sequence listing) that is present in a public database such as GenBankTM at the time of filing of the subject application.
  • An example of such an unmatched target nucleic acid includes, e.g., the nucleic acids co ⁇ esponding to
  • GenBank accession number U55933 and AF312304. Additional such sequences can be identified in GenBank by one of skill.
  • a test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least half as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least half as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 3x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids, such as U55933 or AF312304.
  • Ultra high-stringency hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least 5x as high as that observed for hybridization to any of the unmatched target nucleic acids, such as U55933 or AF312304.
  • a target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least half that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-high stringency conditions.
  • even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx, 20X, 50X, 100X, or even 500X or more as high as that observed for hybridization to any of the unmatched target nucleic acids (U55933) can be identified.
  • a target nucleic acid which hybridizes to a probe under such conditions, with a signal to noise ratio of at least half that of the perfectly matched complementary target nucleic acid is said to bind to the probe under ultra-ultra-high stringency conditions.
  • Target nucleic acids which hybridize to the nucleic acids represented by SEQ DD NO: 1 to SEQ DD NO:48 under high, ultra-high and ultra-ultra high stringency conditions are a feature of the invention.
  • nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.
  • Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical.
  • the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from SEQ JJD NO: 1 to SEQ DD NO:48.
  • the unique subsequence is unique as compared to a nucleic acid co ⁇ esponding to U55933 or any other triazine hydrolase homologue nucleic acid (other than those in the accompanying sequence listing) that is present in a public database such as GenBankTM at the time of filing of the subject application.
  • Such unique subsequences can be determined by aligning any of SEQ DD NO: 1 to SEQ DD NO:48 against the complete set of nucleic acids co ⁇ esponding to known atrazine or triazine hydrolases.
  • Alignment can be performed using the BLAST algorithm set to default parameters. Any unique subsequence is useful, e.g., as a probe to identify the nucleic acids of the invention.
  • the invention includes a polypeptide which comprises a unique subsequence in a polypeptide selected from: SEQ DD NO: 49 to SEQ DD NO: 608.
  • the unique subsequence is unique as compared to a polypeptide co ⁇ esponding U55933, AF312304, or any other triazine hydrolase homologue nucleic acid (other than those in the accompanying sequence listing) that is present in a public database such as GenBankTM at the time of filing of the subject application (the control polypeptides) (note that where the sequence co ⁇ esponds to a non-translated sequence such as a pseudo gene, the co ⁇ esponding polypeptide is generated simply by in silico translation of the nucleic acid sequence into an amino acid sequence, where the reading frame is selected to co ⁇ espond to the reading frame of homologous triazine hydrolase nucleic acids.
  • the invention also provides for target nucleic acids that hybridize under stringent conditions to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from: SEQ DD NO:49 to SEQ DD NO:608, wherein the subsequence is unique as compared to a polypeptide co ⁇ esponding to any of the control polypeptides, e.g., the polypeptide encoded by the nucleic acid represented by U55933, AF312304, or any other triazine hydrolase nucleic acid that is present in a public database such as GenBankTM at the time of filing of the subject application. Unique sequences are determined as noted above.
  • the stringent conditions are selected such that a perfectly complementary oligonucleotide to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10x higher signal to noise ratio than for hybridization of the perfectly complementary oligonucleotide to a control nucleic acid co ⁇ esponding to any of the control polypeptides.
  • Conditions can be selected such that higher ratios of signal to noise are observed in the particular assay which is used, e.g., about 15x, 20x, 30x, 50x or more.
  • the target nucleic acid hybridizes to the unique coding oligonucleotide with at least a 2x higher signal to noise ratio as compared to hybridization of the control nucleic acid to the coding oligonucleotide.
  • higher signal to noise ratios can be selected, e.g., about 5x, lOx, 20x, 30x, 50x or more.
  • the particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radio active label, or the like.
  • the polynucleotides of the invention are optionally used as substrates for a variety of recombination and recursive recombination (e.g., DNA shuffling) reactions and/or other diversity generating reactions, in addition to or concu ⁇ ent with standard cloning methods, to produce triazine hydrolase homologues with desired properties.
  • recombination and recursive recombination e.g., DNA shuffling
  • other diversity generating reactions in addition to or concu ⁇ ent with standard cloning methods, to produce triazine hydrolase homologues with desired properties.
  • a variety of such reactions are known, including those developed by the inventors and their co-workers.
  • nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
  • nucleic acids can be recursively recombined in vivo, e.g., by allowing recombination to occur between nucleic acids in cells.
  • whole cell genome recombination methods can be used in which whole genomes of cells are recombined, optionally including spiking of the genomic recombination mixtures with desired library components such as triazine hydrolase nucleic acids.
  • oligonucleotides co ⁇ esponding to different triazine hydrolases are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which co ⁇ espond to more than one parental nucleic acid, thereby generating new recombined nucleic acids.
  • Oligonucleotides can be made by standard nucleotide addition methods, or can be made by tri-nucleotide synthetic approaches.
  • Fifth, in silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which co ⁇ espond to triazine hydrolases such as those listed in the sequence listing herein.
  • the resulting recombined sequence strings are optionally converted into nucleic acids by synthesis of nucleic acids which co ⁇ espond to the recombined sequences, e.g., in concert with oligonucleotide synthesis/gene reassembly techniques. Any of the preceding general recombination formats are optionally practiced in a reiterative fashion to generate a more diverse set of recombinant nucleic acids.
  • nucleic acids of the invention are optionally recombined (with each other or with related (or even unrelated) nucleic acids) to produce a diverse set of recombinant nucleic acids, including homologous nucleic acids.
  • sequence recombination techniques described herein provide particular advantages in that they provide for recombination between the nucleic acids of SEQ DD NO: 1 to SEQ DD NO:48 or derivatives thereof, in any available format, thereby providing a very fast way of exploring the manner in which different combinations of sequences can affect a desired result.
  • desired results for improved triazine hydrolases include, but are not limited to, the ability to hydrolyze a different substrate, e.g., with a different leaving group or different steric hindrance properties.
  • any nucleic acids which are produced can be selected for a desired activity.
  • this can include testing for and identifying triazine hydrolase activities, by any of the assays in the art.
  • useful properties such as the ability to hydrolyze a variety of substrates with a variety of leaving groups can also be selected for.
  • a variety of triazine hydrolase related (or even unrelated) properties are optionally assayed for, using any available assay.
  • a recombinant nucleic acid produced by recursively recombining one or more polynucleotides of the invention with one or more additional nucleic acid also forms a part of the invention.
  • the one or more additional nucleic acid may include another polynucleotide of the invention; optionally, alternatively, or in addition, the one or more additional nucleic acid can include, e.g., a nucleic acid encoding a naturally-occurring triazine hydrolase or a subsequence thereof, any homologous triazine hydrolase sequence or subsequence thereof, or any triazine hydrolase sequence as found in GenBank or other available literature, or, e.g., any other homologous or non-homologous nucleic acid (certain recombination formats noted above, notably those performed synthetically or in silico, do not require homology for recombination).
  • recombining steps may be performed in vivo, in vitro, or in silico as described in more detail in the references above.
  • a cell containing any resulting recombinant nucleic acid, nucleic acid libraries produced by recursive recombination of the nucleic acids set forth herein, and populations of cells, vectors, viruses, plasmids or the like comprising the library or comprising any recombinant nucleic acid resulting from recombination (or recursive recombination) of a nucleic acid as set forth herein with another such nucleic acid, or an additional nucleic acid.
  • Co ⁇ esponding sequence strings in a database present in a computer system or computer readable medium are a feature of the invention.
  • the invention also includes compositions comprising two or more polynucleotides of the invention (e.g., as substrates for recombination).
  • the composition can comprise a library of recombinant nucleic acids, where the library contains at least 2, 3, 5, 10, 20, or 50 or more nucleic acid species.
  • the nucleic acids are optionally cloned into expression vectors, providing expression libraries.
  • the invention also includes compositions produced by digesting one or more polynucleotide of the invention with a restriction endonuclease, an RNAse, or a DNAse (e.g., as is performed in certain of the recombination formats noted above); and compositions produced by fragmenting or shearing one or more polynucleotide of the invention by mechanical means (e.g., sonication, vortexing, flow based fragmentation, and the like), which can also be used to provide substrates for recombination in the methods above.
  • a restriction endonuclease e.g., an RNAse, or a DNAse
  • compositions produced by fragmenting or shearing one or more polynucleotide of the invention by mechanical means e.g., sonication, vortexing, flow based fragmentation, and the like
  • compositions comprising sets of oligonucleotides co ⁇ esponding to more than one nucleic acid of the invention are useful as recombination substrates and are a feature of the invention.
  • these fragmented, sheared, or oligonucleotide synthesized mixtures are refe ⁇ ed to as fragmented nucleic acid sets.
  • compositions produced by incubating one or more of the fragmented nucleic acid sets in the presence of ribonucleotide- or deoxyribonucleotide triphosphates and a nucleic acid polymerase are also included in the invention.
  • the nucleic acid polymerase may be an RNA polymerase, a DNA polymerase, or an RNA-directed DNA polymerase (e.g., a "reverse transcriptase”); the polymerase can be, e.g. , a thermostable DNA polymerase (such as, VENT, TAQ, or the like).
  • the invention provides isolated or recombinant triazine hydrolase polypeptides, refe ⁇ ed to herein as "triazine hydrolase polypeptides” or simply “triazine hydrolases.”
  • An isolated or recombinant triazine hydrolase polypeptide of the invention includes a polypeptide comprising a sequence selected from SEQ DD
  • amino acid residues in these positions are typically different in the improved hydrolases of the invention as compared to the equivalent position of known, naturally-occurring or recombinant triazine hydrolase sequences, i.e., U55933.
  • the triazine hydrolases of the present invention with variations at these positions exhibit improved activity as compared with atrazine chlorohydrolase U55933 or activity against a novel or alternative substrate.
  • Other unique (as compared to U55933) amino acid residues present in some of the polypeptides of the invention co ⁇ espond to amino acid positions 30, 160, 386, and 465 (nucleic acid positions 89, 478, 1157, and 1395).
  • the invention includes a triazine hydrolase polypeptide comprising at least about 20 or at least about 50 contiguous amino acids of any one of SEQ DD NO:49-608, and one or more amino acid at position 84, 92, 125, 217, 219, 253, 255, 328, and 331 that is unique as compared to U55933 or AF312304, wherein the numbering of the amino acids co ⁇ esponds to that of SEQ DD NO:49.
  • the invention includes a triazine hydrolase polypeptide that is at least about 70% homologous to SEQ DD NO: 49, wherein position number 84 comprises leucine or phenylalanine, position number 92 comprises a leucine, valine or alanine residue, position number 125 comprises glutamic acid, position number 217 comprises threonine, position number 219 comprises threonine, position number 253 comprises leucine or isoleucine, position number 255 comprises glycine or tryptophan, position number 328 comprises aspartic acid or asparagine, and position number 331 comprises serine or cysteine and wherein the polypeptide is unique as compared to atrazine chlorohydrolase (atzA, U55933) or triazine hydrolase (AF312304).
  • position number 84 comprises leucine or phenylalanine
  • position number 92 comprises a leucine, valine or alanine residue
  • position number 125 comprises glutamic acid
  • prefe ⁇ ed polypeptides of the present invention include, but are not limited to, modified versions of atzA (U55933) or AF312304, wherein the modified sequences comprise one or more modification selected from: L 84 , Lc) 2 , D 1 5 , ⁇ -21 7 , P21 9 , L 253 , W 255 , D 328 , and C 331 . All numbering co ⁇ esponds to SEQ DD NO: 49. These polypeptides typically comprise a triazine hydrolase activity, such as activity toward atrazine, atratone, or the like.
  • Example modifications include, the following:
  • V 92 E 1 5 , T 217 , P 2 i 9 , L 53 , G 255 , and S 331 ;
  • V 2 E 125 , 1 217 , P 2 ⁇ 9 , L 2 53, 5 5, and S 331 ;
  • V 92 E 125 , 1 2 ⁇ 7 , P 2 ⁇ , L 53 , G 255 , and S 331 ;
  • V 92 E 125 , T 217 , P 219 , L 253 , G 255 , and C 331 ;
  • F 84 Lg 2j E 12 5, J-2S3, G 255 , D 3 8 , and C 331 ;
  • Prefe ⁇ ed polypeptide sequences showing novel triazine hydrolase activity include, but are not limited to, the following polypeptides represented in Table 5.
  • library members 128, 124, 256, and 252 are prefe ⁇ ed atrazine hydrolases.
  • Prefe ⁇ ed hydrolases for activity toward atratone include library members 121, 113, 125, 117, 377, 369, 57, 49, 381, 373, 61, 53, 313, 305, 317, and 309.
  • Prefe ⁇ ed ametryn hydrolases include library members 378, 377, 382, 314, 381, 313, 379, 383, 370, 121, 318, 317, 369, 125, and 374.
  • Prefe ⁇ ed hydrolases having activity toward amino-atrazine include, but are not limited to, library members 309, 373, 317, 381, 305, 369, 313, and 377.
  • Prefe ⁇ ed prometryn hydrolases include, but are not limited to, library members 498, 502, 506, 482, 497, 510, 501, 505, and 466.
  • Library members 252, 244, 508, 256, 500, 248, 512, and 504 comprise prefe ⁇ ed hydrolases for propazine substrates and library members 505, 509, 507, 506, 497, 249, and 441 are prefe ⁇ ed for activity against amino-propazine.
  • NME-propazine activity is represented by prefe ⁇ ed library members 477, 473, 509, 505, 469, 465, 501, 506, and 510 in Table 5 and prefe ⁇ ed NME-atrazine hydrolases include library members 377, 381, 505, 369, 509, 373, 121, 313, 125, 345, 317, and 349.
  • the substrate activity column in Table 5 indicates prefe ⁇ ed embodiments and is not exclusive.
  • the polypeptides may be produced by direct peptide synthesis using solid- phase techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) /. Am. Chem. Soc. 85:2149-2154). Peptide synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer. For example, subsequences may be chemically synthesized separately and combined using chemical methods to provide full-length triazine hydrolases.
  • a triazine hydrolase polypeptide of the invention is used to produce antibodies which have, e.g., diagnostic and therapeutic uses, e.g., related to the activity, distribution, and expression of triazine hydrolases.
  • Antibodies to triazine hydrolases of the invention may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which block receptor binding, are especially prefe ⁇ ed for therapeutic use.
  • Triazine hydrolase polypeptides for antibody induction do not require biological activity; however, the polypeptide or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least 10 amino acids, preferably at least 15 or 20 amino acids. Short stretches of a triazine hydrolase polypeptide may be fused with another protein, such as keyhole limpet hemocyanin, and antibody produced against the chimeric molecule. Methods of producing polyclonal and monoclonal antibodies are known to those of skill in the art, and many antibodies are available. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about 0.1 ⁇ M, preferably at least about 0.01 ⁇ M or better, and most typically and preferably, 0.001 ⁇ M or better.
  • Triazine hydrolase polypeptides of the present invention include conservatively modified variations of the sequences disclosed herein as SEQ DD
  • Such conservatively modified variations comprise substitutions, additions or deletions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than about 5%, more typically less than about 4%, 2%, or 1%) in any of SEQ JJD NO:49 to SEQ JJD NO:608.
  • a conservatively modified variation (e.g., deletion) of the 474 amino acid polypeptide identified herein as SEQ JJD NO:49 will have a length of at least about 450 amino acids, preferably at least about 455 amino acids, more preferably at least about 465 amino acids, and still more preferably at least about 470 amino acids, co ⁇ esponding to a deletion of less than about 5%, 4%, 2% or 1% of the polypeptide sequence.
  • a conservatively modified variation e.g., a "conservatively substituted variation” of the polypeptide identified herein as SEQ JJD NO:49 will contain "conservative substitutions", according to the six substitution groups set forth in Table 2 (supra), in up to about 23 residues (i.e., less than about 5%) of the 474 amino acid polypeptide.
  • the triazine hydrolase polypeptide sequences of the invention can be present as part of larger polypeptide sequences such as occur upon the addition of one or more domains for purification of the protein (e.g., poly his segments, FLAG tag segments, etc.), e.g., where the additional functional domains have little or no effect on the activity of the triazine hydrolase portion of the protein, or where the additional domains can be removed by post synthesis processing steps such as by treatment with a protease.
  • domains for purification of the protein e.g., poly his segments, FLAG tag segments, etc.
  • polypeptides of the invention comprise at least about 30, or at least about 50, at least about 70, at least about 100, at least about 120, at least about 150, or at least about 155 contiguous amino acid residues of any one of SEQ DD NO:49-608.
  • polypeptides of the invention provide a variety of new polypeptide sequences as compared to other triazine hydrolases, the polypeptides also provide new structural features which can be recognized, e.g., in immunological assays.
  • the generation of antisera which specifically binds the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are a feature of the invention.
  • the invention includes triazine hydrolase proteins that specifically bind to or that are specifically immunoreactive with an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of SEQ DD NO: SEQ DD NO: 49 to SEQ JJD NO: 608.
  • the antibody or antisera is subtracted with available triazine hydrolases, such as that represented at GenBank accession numbers U55933 (a control triazine hydrolase nucleic acid). Where the accession number co ⁇ esponds to a nucleic acid, a polypeptide encoded by the nucleic acid is generated and used for antibody/antisera subtraction purposes.
  • nucleic acid co ⁇ esponds to a non- coding sequence, e.g., a pseudo gene
  • an amino acid which co ⁇ esponds to the reading frame of the nucleic acid is generated (e.g., synthetically), or is minimally modified to include a start codon for recombinant production.
  • the immunoassay uses a polyclonal antiserum which was raised against one or more polypeptide comprising one or more of the sequences co ⁇ esponding to one or more of : SEQ DD NO:49 to SEQ DD NO: 608 or a ' substantial subsequence thereof (i.e., at least about 30% of the full length sequence provided).
  • the full set of potential polypeptide immunogens derived from SEQ DD NO:49 to SEQ DD NO: 608 are collectively refe ⁇ ed to below as "the immunogenic polypeptides.”
  • the resulting antisera is optionally selected to have low cross- reactivity against the control triazine hydrolases other known triazine hydrolases and any such cross-reactivity is removed by immunoabsorbtion with one or more of the control triazine hydrolases, such as atrazine chlorohydrolase, prior to use of the polyclonal antiserum in the immunoassay.
  • one or more of the immunogenic polypeptides is produced and purified as described herein.
  • recombinant protein may be produced in a mammalian cell line.
  • An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic protein(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see, Harlow and Lane (1988) Antibodies, A
  • one or more synthetic or recombinant polypeptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
  • Polyclonal sera are collected and titered against the immunogenic polypeptide in an immunoassay, for example, a solid phase immunoassay with one or more of the immunogenic proteins immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 6 or greater are selected, pooled and subtracted with the control triazine hydrolase polypeptides to produce subtracted pooled titered polyclonal antisera.
  • the subtracted pooled titered polyclonal antisera are tested for cross reactivity against the control triazine hydrolases.
  • Preferably at least two of the immunogenic triazine hydrolases are used in this determination, preferably in conjunction with at least two control hydrolase homologues, to identify antibodies which are specifically bound by the immunogenic protein(s).
  • discriminatory binding conditions are determined for the subtracted titered polyclonal antisera which result in at least about a 5-10 fold higher signal to noise ratio for binding of the titered polyclonal antisera to the immunogenic triazine hydrolases as compared to binding to the control triazine hydrolases. That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or non-fat dry milk, or by adjusting salt conditions, temperature, or the like. These binding conditions are used in subsequent assays for determining whether a test polypeptide is specifically bound by the pooled subtracted polyclonal antisera.
  • test polypeptides which show at least a 2-5x higher signal to noise ratio than the control polypeptides under discriminatory binding conditions, and at least about a one half signal to noise ratio as compared to the immunogenic polypeptide(s), shares substantial structural similarity with the immunogenic polypeptide as compared to known triazine hydrolases, and is, therefore a polypeptide of the invention.
  • immunoassays in the competitive binding format are used for detection of a test polypeptide.
  • cross-reacting antibodies are removed from the pooled antisera mixture by immunoabsorbtion with the control triazine hydrolase polypeptides.
  • the immunogenic polypeptide(s) are then immobilized to a solid support which is exposed to the subtracted pooled antisera.
  • Test proteins are added to the assay to compete for binding to the pooled subtracted antisera.
  • test protein(s) The ability of the test protein(s) to compete for binding to the pooled subtracted antisera as compared to the immobilized protein(s) is compared to the ability of the immunogenic polypeptide(s) added to the assay to compete for binding (the immunogenic polypeptides compete effectively with the immobilized immunogenic polypeptides for binding to the pooled antisera).
  • the percent cross- reactivity for the test proteins is calculated, using standard calculations.
  • the ability of the control proteins to compete for binding to the pooled subtracted antisera is determined as compared to the ability of the immunogenic polypeptide(s) to compete for binding to the antisera.
  • percent cross-reactivity for the control polypeptides is calculated, using standard calculations. Where the percent cross-reactivity is at least 5-10x as high for the test polypeptides, the test polypeptides are said to specifically bind the pooled subtracted antisera.
  • the immunoabsorbed and pooled antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide to the immunogenic polypeptide(s).
  • the two polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the subtracted antisera to the immobilized protein is determined using standard techniques. If the amount of the test polypeptide required is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to specifically bind to an antibody generated to the immunogenic protein, provided the amount is at least about 5-10x as high as for a control polypeptide.
  • the pooled antisera is optionally fully immunabsorbed with the immunogenic polypeptide(s) (rather than the control polypeptides) until little or no binding of the resulting immunogenic polypeptide subtracted pooled antisera to the immunogenic polypeptide(s) used in the immunoabsorbtion is detectable.
  • This fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If little or no reactivity is observed (i.e., no more than 2x the signal to noise ratio observed for binding of the fully immunoabsorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
  • the triazine hydrolases of the invention are optionally used in compositions (in vivo or in vitro) to serve as decontamination or cleaning solutions for water or soil contaminated with atrazine or another triazine derivative, such as aminotriazine, atrazine, atratone, N- methylatiazine, ametryn, aminopropazine, propazine, prometon, N-methylpropazine, prometryn, aminomorphazine, morphazine, morphatryn, morphaton, or N- methylmorphazine, or the like (as described above).
  • atrazine or another triazine derivative such as aminotriazine, atrazine, atratone, N- methylatiazine, ametryn, aminopropazine, propazine, prometon, N-methylpropazine, prometryn, aminomorphazine, morphazine, morphatryn, morphaton, or N- methylmorphazine, or the
  • the polypeptides presented herein provide hydrolases that degrade novel substrates in comparison to wild-type atrazine chlorohydrolase.
  • the hydrolases of the invention are optionally used to remove alternate leaving groups from triazine derivatives.
  • NH 2 , Cl or other halogens, 0-CH 3 , -NHCH 3 , -SCH 3 , or the like are optionally removed from a triazine derivative by one or more of the hydrolases provided herein.
  • substrates with greater or lesser steric hindrance are also degraded by one or more the hydrolases provided herein. Examples of substrate compounds are provided as follows:
  • Ri and R 3 each independently comprise an amino group, i.e., -NH 2 , or a substituted linear, branched , or cyclic amino group.
  • Ri and R 3 are each independently a lower-alkyl-substituted amino group or a morpholino group.
  • the term "lower alkyl” refers to a C ⁇ - 6 alkyl. More typically, Ri and R 3 are each independently -NH(C 2 H 5 ), -NHCH(CH 3 ) 2 ,
  • R 3 is -NHCH(CH 3 ) 2
  • R2 is an amino group, i.e., -NH 2j or an optionally substituted amino group, e.g., -NRH or -NRR', a halo, a lower alkoxy, or - S-R, where R and R' are each independently a lower alkyl group.
  • R 2 is - NH 2 , -X, wherein X is a halogen such as Cl, -OCH 3 , -NH(CH 3 ), or -S-CH 3 .
  • a larger Ri and/or R 3 group leads to greater steric hindrance.
  • Tables 3 and 4 provide data illustrating the use of the novel polypeptides of the invention against substrates that cannot be degraded by other triazine hydrolases including substrates such as ametryn, N-methylatrazine, prometryn, N-methylpropazine, prometon, and the like.
  • the present invention provides for the use of the novel triazine hydrolases of the invention in decontamination solutions, as well as such compositions containing the mutant hydrolase enzymes.
  • Such solutions in principle have any physical form, e.g., tablets, solutions, cell cultures, etc.
  • cells transformed with the recombinant genes for triazine hydrolases provided herein are used to express the hydrolases. The cells are mixed with soil, or water for decontamination where the expressed enzyme degrades or hydrolyzes the triazine derivative, thus purifying the soil or water sample.
  • the present invention provides computers, computer readable media and integrated systems comprising character strings co ⁇ esponding to the sequence information herein for the polypeptides and nucleic acids herein, including, e.g., those sequences listed herein and the various silent substitutions and conservative substitutions thereof.
  • GOs genetic algorithms
  • homology determination methods have been designed for comparative analysis of sequences of biopolymers, for spell-checking in word processing, and for data retrieval from various databases.
  • models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings co ⁇ esponding to the sequences herein (e.g., word-processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).
  • An example of a software package with GOs for calculating sequence similarity is BLAST, which can be adapted to the present invention by inputting character strings co ⁇ esponding to the sequences herein.
  • standard desktop applications such as word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM) and database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs such as Microsoft AccessTM or ParadoxTM) can be adapted to the present invention by inputting a character string co ⁇ esponding to the triazine hydrolases of the invention (either nucleic acids or proteins, or both).
  • the integrated systems can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system) to manipulate strings of characters.
  • a user interface e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system
  • Integrated systems for analysis in the present invention typically include a digital computer with GO software for aligning sequences, as well as data sets entered into the software system comprising any of the sequences herein.
  • the computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, OS2TM WINDOWSTM WJ-NDOWS NTTM, WTNDOWS95TM, WINDOWS98TM LINUX based machine, a MACINTOSHTM, Power PC, or a UNIX based (e.g., SUNTM work station) machine) or other commercially common computer which is known to one of skill.
  • Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, Fortran, Basic, Java, or the like.
  • Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display), or others.
  • Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others.
  • the box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements.
  • Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.
  • the computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
  • the software then converts these instructions to appropriate language for instructing the operation of the fluid direction and transport controller to cany out the desired operation.
  • the software can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of a sequences herein) or other operations which occur downstream from an alignment or other operation performed using a character string co ⁇ esponding to a sequence herein.
  • kits embodying the methods, composition, systems and apparatus herein optionally comprise one or more of the following: (1) an apparatus, system, system component or apparatus component as described herein; (2) instructions for practicing the methods described herein, and/or for operating the apparatus or apparatus components herein and/or for using the compositions herein; (3) one or more triazine hydrolase composition or component; (4) a container for holding components or compositions, and, (5) packaging materials.
  • the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.

Abstract

L'invention concerne de nouvelles hydrolases de triazine (acides nucléiques et protéines). L'invention concerne également des compositions comprenant ces nouvelles protéines et/ou gènes, des cellules de recombinaison, des méthodes de brassage dans lesquelles sont utilisées les nouvelles hydrolases de triazine, des anticorps des nouvelles hydrolases de triyzine, et des méthodes d'utilisation des hydrolases de triazine.
PCT/US2001/006654 2000-02-29 2001-02-28 Enzymes de degradation de triazine WO2001064912A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP01916339A EP1268815A2 (fr) 2000-02-29 2001-02-28 Enzymes de degradation de triazine
AU2001243376A AU2001243376A1 (en) 2000-02-29 2001-02-28 Triazine degrading enzymes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18580900P 2000-02-29 2000-02-29
US60/185,809 2000-02-29

Publications (2)

Publication Number Publication Date
WO2001064912A2 true WO2001064912A2 (fr) 2001-09-07
WO2001064912A3 WO2001064912A3 (fr) 2002-05-10

Family

ID=22682531

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/006654 WO2001064912A2 (fr) 2000-02-29 2001-02-28 Enzymes de degradation de triazine

Country Status (4)

Country Link
US (1) US20020155571A1 (fr)
EP (1) EP1268815A2 (fr)
AU (1) AU2001243376A1 (fr)
WO (1) WO2001064912A2 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009076711A1 (fr) * 2007-12-19 2009-06-25 Sugar Industry Innovation Pty Ltd Enzymes et procédés pour dégrader les s-triazines chlorées
WO2015075048A1 (fr) * 2013-11-21 2015-05-28 Dsm Ip Assets B.V. Procédé de préparation de l'ammeline
US9228223B2 (en) 2007-02-12 2016-01-05 Codexis, Inc. Structure-activity relationships
US20160355667A1 (en) * 2013-11-21 2016-12-08 Dsm Ip Assets B.V. Flame-retardant polyamide composition
CN107426979A (zh) * 2015-01-21 2017-12-01 巴斯夫欧洲公司 具有增加的除草剂耐受性的植物
CN109688807A (zh) * 2016-07-15 2019-04-26 巴斯夫欧洲公司 具有增加的除草剂耐受性的植物

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112020013929A2 (pt) * 2018-01-17 2020-12-01 Basf Se plantas ou partes da planta, semente, células vegetais, produto vegetal, progênie ou planta descendente, métodos para controlar ervas daninhas, para produzir uma planta e para produzir uma planta descendente, molécula de ácido nucleico, cassete de expressão, vetor, polipeptídeo, método para produzir um produto vegetal e produto vegetal

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5605793A (en) * 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
WO1997015675A1 (fr) * 1995-10-23 1997-05-01 Regents Of The University Of Minnesota Molecule d'adn et proteine isolees et purifiees permettant la degradation des composes de triazine
WO1998007830A2 (fr) * 1996-08-22 1998-02-26 The Institute For Genomic Research SEQUENCE GENOMIQUE COMPLETE D'UNE ARCHEOBACTERIE METHANOGENE, $i(METHANOCOCCUS JANNASCHII)
WO1998031816A1 (fr) * 1997-01-17 1998-07-23 Regents Of The University Of Minnesota Molecules d'adn et proteine presentant une capacite amelioree de degradation des composes de triazine

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6369299B1 (en) * 1999-06-10 2002-04-09 Regents Of The University Of Minnesota Transgenic plants expressing bacterial atrazine degrading gene AtzA

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5605793A (en) * 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US5830721A (en) * 1994-02-17 1998-11-03 Affymax Technologies N.V. DNA mutagenesis by random fragmentation and reassembly
WO1997015675A1 (fr) * 1995-10-23 1997-05-01 Regents Of The University Of Minnesota Molecule d'adn et proteine isolees et purifiees permettant la degradation des composes de triazine
WO1998007830A2 (fr) * 1996-08-22 1998-02-26 The Institute For Genomic Research SEQUENCE GENOMIQUE COMPLETE D'UNE ARCHEOBACTERIE METHANOGENE, $i(METHANOCOCCUS JANNASCHII)
WO1998031816A1 (fr) * 1997-01-17 1998-07-23 Regents Of The University Of Minnesota Molecules d'adn et proteine presentant une capacite amelioree de degradation des composes de triazine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL S F ET AL: "BASIL LOCAL ALIGNMENT SEARCH TOOL" JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 215, 1990, pages 403-410, XP000604562 ISSN: 0022-2836 *
DATABASE EM_PRO [Online] EMBL; 17 August 1996 (1996-08-17) DE SOUZA ET AL.: "Pseudomonas ADP atrazine chlorohydrolase (atzA) gene, complete cds." retrieved from EBI, accession no. PAU55933 Database accession no. U55933 XP002179308 cited in the application -& DATABASE SWALL [Online] 6 September 2001 (2001-09-06) DE SOUZA ET AL.: "Atrazine chlorohydrolase" retrieved from EBI, accession no. AAK50270 Database accession no. AAK50270 XP002179309 -& DE SOUZA ET AL.: "Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, no. 9, September 1995 (1995-09), pages 3373-3378, XP000616901 cited in the application *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9228223B2 (en) 2007-02-12 2016-01-05 Codexis, Inc. Structure-activity relationships
WO2009076711A1 (fr) * 2007-12-19 2009-06-25 Sugar Industry Innovation Pty Ltd Enzymes et procédés pour dégrader les s-triazines chlorées
US10584230B2 (en) * 2013-11-21 2020-03-10 Dsm Ip Assets B.V. Flame-retardant polyamide composition
WO2015075048A1 (fr) * 2013-11-21 2015-05-28 Dsm Ip Assets B.V. Procédé de préparation de l'ammeline
CN105745328A (zh) * 2013-11-21 2016-07-06 帝斯曼知识产权资产管理有限公司 制备三聚氰酸二酰胺的方法
US20160355667A1 (en) * 2013-11-21 2016-12-08 Dsm Ip Assets B.V. Flame-retardant polyamide composition
US10590447B2 (en) 2013-11-21 2020-03-17 Dsm Ip Assets B.V. Process for the preparation of ammeline
EP3247200A4 (fr) * 2015-01-21 2018-06-13 Basf Se Plantes présentant une tolérance accrue aux herbicides
US10538782B2 (en) 2015-01-21 2020-01-21 Basf Se Plants having increased tolerance to herbicides
CN107426979A (zh) * 2015-01-21 2017-12-01 巴斯夫欧洲公司 具有增加的除草剂耐受性的植物
AU2016209901B2 (en) * 2015-01-21 2021-12-02 Basf Se Plants having increased tolerance to herbicides
EP3484278A4 (fr) * 2016-07-15 2020-02-19 Basf Se Plantes présentant une tolérance accrue aux herbicides
CN109688807A (zh) * 2016-07-15 2019-04-26 巴斯夫欧洲公司 具有增加的除草剂耐受性的植物
US11499162B2 (en) 2016-07-15 2022-11-15 Basf Se Plants having increased tolerance to herbicides
AU2017294685B2 (en) * 2016-07-15 2023-10-26 Basf Se Plants having increased tolerance to herbicides

Also Published As

Publication number Publication date
WO2001064912A3 (fr) 2002-05-10
EP1268815A2 (fr) 2003-01-02
AU2001243376A1 (en) 2001-09-12
US20020155571A1 (en) 2002-10-24

Similar Documents

Publication Publication Date Title
US7220566B2 (en) Subtilisin variants
AU2001249811A1 (en) Subtilisin variants
US20090298123A1 (en) Novel Lipase Genes
WO2002006457A2 (fr) Genes de lipase
CA2553734A1 (fr) Adn-polymerase arn-dependante tiree de geobacillus stearothermophilus
CN111437384A (zh) 用于预防covid-19的蝙蝠源性冠状病毒疫苗
EP1268815A2 (fr) Enzymes de degradation de triazine
Cortez et al. Genetic organisation of Iris yellow spot virus M RNA: indications for functional homology between the G (C) glycoproteins of tospoviruses and animal-infecting bunyaviruses
Ben-Shaul et al. Genomic diversity among populations of two citrus viroids from different graft-transmissible dwarfing complexes in Israel
Wang et al. Complete genomic sequence analyses of Turnip mosaic virus basal-BR isolates from China
Roy et al. Genotype classification and molecular evidence for the presence of mixed infections in Indian Citrus tristeza virus isolates
US7611897B2 (en) AP1 amine oxidase variants
EP1572861A2 (fr) Variants d'amine oxydase ap1
Mu¨ ller et al. Identification of a thymidylate synthase gene within the genome of Chilo iridescent virus
US6835538B1 (en) Method of genetic modification of a wild type viral sequence
WO2000001718A2 (fr) Domaine catalytique ns4a et ns3 de l'hepatite c
WO2005003289A2 (fr) Variants de l'amine oxydase ap1
EP1619246A1 (fr) ARN-polymerases ARN-dépendante de coronavirus et leur utilisations en biologie moléculaire et pour le criblage de médicaments
CA2708609A1 (fr) Modification genetique de zymogene pour la toxicite conditionnelle
JP2001514505A (ja) ニューモシスティス・カリニのプロテアーゼをコードするdna
Takács et al. Phenotype and Genetic Heterogeneity of Some Tuber Necrosis Isolates of Potato virus Y (PVYNTN)
WO1999055848A2 (fr) Clonage et expression de plasmepsines recombinantes
ZA200500910B (en) AP1 Amine oxidase variants

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2001916339

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2001916339

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2001916339

Country of ref document: EP